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This invention relates generally to medical diagnostics and, more specifically, to a method of diagnosing and staging malignant tumors using an improved method of immunohistochemistry to determine the concentration and distribution of a cytoplasmic tumor marker associated with malignant tumors.
The inventor has previously identified, by cDNA cloning, a gene denoted Metallopanstimulin (MPS-1). The novel DNA sequence which encodes the MPS-1 is disclosed in U.S. Pat. No. 5,243,041 (Re. 35,585), issued Sep. 7, 1993, the disclosure of which is hereby incorporated by reference. The recombinant MPS-1 protein and chemical derivatives can be used to generate polyclonal and monoclonal anti-MPS-1 antibodies. The MPS-1 gene is expressed at high levels in numerous human carcinoma cell lines such as prostate, breast, brain, lung, and particularly melanomas. It is also expressed in human hematological malignancies. MPS-1 is a multifunctional S27 ribosomal protein that is involved in cellular proliferation and oncogenesis in numerous human neoplasms. The MPS-1 gene is expressed at high levels in numerous human carcinoma cell lines such as breast, prostate, colon, brain, lung and melanoma. Table IA indicates the presence of MPS-1 mRNA in cultured human malignant cell lines and in peripheral blood of human patients with hematological malignancies. Cytoplasmic MPS-1 is of particular interest because of the proliferation of cytoplasmic mRNA in proliferative diseases, including cancers. Table 1B indicates the presence of MPS-1 mRNA in human hematological malignancies.
Table 2 illustrates the results of tests conducted to detect MPS-1 mRNA and protein in human tissues.
Table 2 should be read as follows:
Signals: (xe2x88x92) negative, (+) weakly positive, (++) positive, and (+++) strongly positive. The stainings recorded refer to that of the cancer cells, since the stroma cells were not significantly stained. Note that although normal tissues are listed as (xe2x88x92), they showed staining (+ to +++) only in areas of normal cell proliferation. ND: Not done. Note that (i) vulvar melanoma, and breast inflammatory carcinoma had the highest levels of MPS-1 mRNA and protein detected; by Northern blot analysis the MPS-1 mRNA levels in these tissues were  greater than 80-fold normal levels; and (ii) lichen sclerosus atrophicus, a rare condition characterized by extremely low proliferation rates was negative for MPS-1 mRNA and protein in the usual areas of cell multiplication; these observations are highlighted by bold characters.
The inventor has determined that there is a high correlation between a patient""s survival rate and the level of cytoplasmic MPS-1 observed. That is, the patient""s survival rate decreases in proportion to increased concentration distribution of cytoplasmic MPS-1 detected. Hence, the method of the present invention is useful as a prognostic indicator of disease progress and survival.
The MPS-1 mRNA was detected using biotynilated single-stranded anti-sense DNA probe. The MPS-1 protein was detected by immunohistochemical staining using anti-peptide A antibodies. As can be observed, there is an excellent correlation between the concentration and distribution of MPS-1 mRNA and protein expression. In contrast, the MPS-1 gene is expressed at low levels in normal cells. The results of experiments indicated that the MPS-1 antigen is a ubiquitous tumor marker which is useful in detection, prognosis and management of various types of neoplastic conditions, particularly when the concentration and distribution of the protein is detected in the cytoplasm, as recently discovered.
Although the detection and management of all forms of cancer is desirable, the detection of malignant melanoma is particularly challenging to the clinician. Often benign lesions are difficult to distinguish from malignant lesions. It is imperative, however, that cancers such as malignant melanoma, breast cancer and prostate be detected early and reliably to improve survival rates.
Recently, immunohistochemical studies were conducted to examine the expression of MPS-1 protein in various types of benign and malignant melanocytic lesions. Protein antigen, detected with anti MPS-1 antibodies was found in both benign and malignant melanocytic lesions. The anti-MPS-1 antibodies directed to the N-terminal portion of the molecule are most useful. In benign lesions, the staining was weak and in a gradient, the most superficial cells with nesting growth patterns were positive, particularly those within the epidermis. The stain intensity decreased as the melanocytes were located deeper in the dermis. Practically speaking, only type A melanocytes stain positive while the B and C types are negative.
Recurrent melanocytic nevi were also studied. MPS-1 was nearly negative in the original untreated nevi. In the recurrent lesions, the regenerating epidermal and dermal melanocytic components were intensely and evenly stained. These findings were very similar to those seen in melanomas. These changes are an example of intense activation of the newly formed melanocyte population and not a sign of malignant transformation.
It is of interest to note that scar tissue generates large amounts of growth factors. Thus, growth factors may be responsible for both activation of melanocytic cells and intense expression of MPS-1 observed in the incomplete biopsy. It will be appreciated that the histological features of these recurrent nevi were indistinguishable from melanomas, a phenomenon that often confounds the diagnostician. The correct diagnosis was made by reviewing the original melanocytic nevus.
In three of the Spitz""s nevi studied, two were weakly positive or negative. Interestingly, those lesions occurred in adults. The third lesion was strongly positive and occurred in a two year old child. The most interesting findings were seen in congenital nevi, These lesions have a similar gradient staining as seen in regular nevi but also had discrete MPS-1 positive nodules contrasting with negative ones as seen in melanomas. These findings reveal a heterogeneous (clonal?) cell population and reflect, perhaps, an unstable cell population. This is of interest in view of the fact that congenital nevi are at times precursors of melanoma.
In melanomas, the staining patterns are more complex. While some melanomas stain evenly positive, others have remarkable variable expression of MPS-1. This seems to correlate, to some extent, with intralesional transformation. The variability is so pronounced that some cells stain intensely positive in nests of cells staining moderately positive. The scattered melanocytes migrating to the upper layers of the epidermis usually are intensely positive. Curiously, metastatic melanoma to lymph nodes shows only faint positivity in the limited sampling studied. A single example of melanoma metastatic to the skin was evenly and intensely positive in spite of its seemingly well differentiated, almost nevoid appearance. No gradient staining was present, as it should have been in the case of a benign nevus. Thus, the inventor has determined that the novel test described and claimed herein has widespread application in the diagnosis of melanocytic lesions of the skin (Santa Cruz et. al Differential expression of Metallopanstimulin/S27 ribosomal protein in melanocytic lesions of the skin, J. Cutan Pathol 1997; 24:533-542).
Macrophages in and around the area are intensely positive with a coarse, granular cytoplasmic pattern. Macrophages present in less intensely stained areas had less MPS-1 content than those located in strongly stained areas. This was particularly true in nevi in which macrophages were rare or non-existent. This finding tends to correlate with the near absence of apoptosis in nevi. On the other hand, the presence of MPS-1 in macrophages of melanomas suggests direct phagocytosis of melanoma cell debris following apoptosis, a common phenomenon. Some melanocytes in melanomas show individual cells with similar patterns, supporting the concept of phagocytosis by melanoma cells.
MPS-1 also stains reactive acanthotic epidermis, sebaceous glands and the secretory portion of the sweat glands. Hair follicles also are positive in a gradient, with the more mitotically active cells of the matrix staining the strongest. Basal cell carcinomas show even moderate positivity. As the above discussion points out, MPS-1 is an extremely useful marker for melanocytic lesions at the immunohistological level, providing important clues in the biological nature of melanocytic tumors not obtainable by other methods. Also, as stated above, the MPS-1 is a useful prognosticator of patient survival.
It was suggested in Example 12 of U.S. Pat. No. 5,243,041, col. 35, line 14, that antibodies to MPS-1 bind to melanoma cells. Furthermore, it was suggested therein that antibodies that bind to MPS-1 may have diagnostic uses. It also will be appreciated that Example 12 contemplates using antibodies to the MPS-1 protein prepared by the method of Example 11. While the examples listed work well for their intended purposes, which was to show a gross correlation between MPS-1 and cancer and to demonstrate the potential utility of MPS-1 as a tumor marker, the procedures of Example 12, in addition to being generally highly complex, were experimental and lacked any immediate practical application for the clinician.
More importantly, the procedures disclosed in the ""041 patent, as well as in the inventor""s paper (Xynos et al; Anticancer Res. 14:773-786 1994) do not have the widespread clinical applicability of the present invention. First, the procedure disclosed in the ""041 patent and in Xynos et al detects the presence of MPS-1 protein in nuclear material. As set out above, this test had little practical application and was useful to demonstrate that MPS-1 could be considered a ubiquitous tumor marker. The inventor subsequently determined there is little correlation between nuclear staining intensities and elevated MPS-1 levels associated with cancer. Thus, the original techniques had little diagnostic or predictive value. Because the prior art tests detect the presence of MPS-1 proteins in the nucleus, the number of false positive results are elevated. This is because MPS-1 proteins may be found in the nucleus, even in non-malignant cells, and because the prior art methods resulted in a loss of cytoplasmic MPS-1 from the cell membrane. The prior art methods included the use of a detergent, saponin, caused a xe2x80x9cwashing outxe2x80x9d of cytoplasmic MPS-1 and the concomitant staining of nuclear proteins. The chromogenic reaction described in the patent at col. 33, lines 40-43, included the use of Naphthol phosphate/Fast Red TR, which penetrated into the nuclear proteins and resulted in staining of the nuclear proteins. The staining of the nuclear proteins in the prior art methods make distinguishing between normal cells and early cancers very difficult. The inventor later discovered that in malignancies there is elevated MPS-1 protein in the cytoplasm due to the elevated levels of proteins having a recognizable N-terminal portion produced as a ribosomal protein. Moreover, he determined that there is an increase in ribosomal MPS-1 proteins in the cytoplasm that is related to the stage of the cancer. The methods of the present invention conserve cytoplasmic MPS-1 which eliminates the false positive or non-quantifiable detection of nuclear MPS-1 of the old methods. Determining the level of cytoplasmic, i.e. ribosomal MPS-1, is the key to a detecting and staging cancers and the prediction of disease free survival. Thus, the present invention can be used to determine prognosis based upon the amount, i.e. concentration and distribution, of cytoplasmic MPS-1 protein detected.
Furthermore, the procedures outlined in Example 12 are not easily or cost-effective practiced by a diagnostician, in the hospital or in the medical office, to test a tissue sample. I have devised a low cost, simple to use method and kit for practicing the method that takes advantage of the fact that MPS-1 provides a useful marker, but does so in a way not shown or suggested by the U.S. Pat. No. 5,243,041, or any other reference known to the inventor. In addition, the use of a poorly visualized dye as described in Example 12 of the ""041 with melanoma produced only a slightly different brown rendering interpretation of the results extremely difficult.
In summary, the prior art immunohistochemistry procedures lack specificity. The sensitivity is low and the procedures have relatively poor predictive functions. The prior art tests also have high background staining which results in difficult differentiation. In addition to proving the efficacy of the novel immunhistochemistry techniques of the present invention as applied to melanoma, the inventor has demonstrated that the new method of detecting Metallopanstimulin expression in stages I and II breast cancer correlates with clinical and pathological factors. The prior art immunohistochemistry techniques disclosed by the inventor were not satisfactory for detecting breast cancers. The differential expression of MPS-1 in Stage I and Stage II were difficult to analyze because the perceived differences between stained tissue and background were minimal and, because of the permeability of the cells, nuclear protein staining obfuscated the importance of cytoplasmic MPS-1. Furthermore, there is a clinical relationship between the differential expression of Metallopanstimulin in hepatic regeneration and liver oncogenesis. The expression of the Metallopanstimulin (MPS-1) in the cytoplasm in breast and liver disease was studied in tissue samples by immunohistochemistry of the present invention using specific anti-MPS-1 antibodies. The concentration and distribution of cytoplasmic MPS-1 correlates well with disease free survival this provide a reliable prognostic tool.
It is among the several objects of the invention to provide a novel method of performing immunohistochemical staining of a tissue sample to detect cancer cells.
It is also among the several objects of the invention to provide a simple method of performing immunohistochemical staining of a tissue sample to detect the expression of Metallopanstimulin (MPS-1) in cancer cells.
It is another object of the invention to provide a such a method that avoids loss of cytoplasmic Metallopanstimulin.
It is another object of the invention to provide a such a method that allows the detection of the Metallopanstimulin in the cytoplasmic region.
Another object of the invention to provide a such a method that allows the quantification of the concentration and distribution of Metallopanstimulin in the cytoplasmic region
It is another object of the invention to provide a such a method that allows the detection of the Metallopanstimulin in the cytoplasmic region so as to avoid false positives associated with nuclear staining.
Another object of the invention is to provide a method of determining the concentration and distribution of the Metallopanstimulin in the cytoplasmic region of suspected malignant cells to allow quantification, grading or staging of the disease.
Another object of the invention is to provide such a method that employs antibodies directed to the N-terminal portion of the MPS-1 protein.
Another object of the invention is to provide a method of performing immunohistochemical staining wherein the dye renders the Metallopanstimulin easy to visualize.
It is another object of the invention to provide a simple method of performing immunohistochemical staining of a tissue sample using antibodies prepared by using synthetic peptides with partial MPS-1 sequences.
It is another object of the present invention to provide a method of performing immunohistochemical staining of a tissue sample that can be easily performed by a diagnostician in the hospital or office setting.
Still another object of the present invention is to provide the materials needed to perform the novel immunohistochemical staining technique in a simple to use, reasonably priced kit.
In accordance with the invention, a method of performing immunohistochemical staining on a tissue sample and the materials for doing the same are provided. The method is directed to the detection of MPS-1 proteins in the cytoplasm with antibodies directed to the N-terminal portion of the MPS-1 protein. Staining techniques allows the visualization of antibody-protein complexes. The antibody-protein complexes can be quantified to allow gradation of the malignancy. The method avoids the loss of cytoplasmic proteins and avoids which can cause false positive readings, unquantifiable results, and artifacts.
The method consists of preparing a sample holder, which in the embodiment illustrated is a glass slide, with a reagent bonding material; collecting the tissue sample; fixing the tissue sample; embedding the sample in paraffin; deparaffinizing the tissue; preparing the tissue for staining; incubating the slide with a stain blocking agent; incubating the tissue with a primary anti-Peptide A antibody produced in accordance with the procedures described in U.S. Pat. No. 5,243,041 (Re. 35, 585); incubating the sample with red chromogen stain; rinsing the sample; dipping the slide in a standard bluing solution; mounting the slide for reading. The materials for performing the above steps are provided in a convenient, reasonably priced kit.